Thermostable Group II Intron Reverse Transcriptase for Structured RNA Sequencing
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Solution Overview
Problem
Current RNA-seq methods are unable to obtain full-length end-to-end sequence reads of structured spliceosomal intron RNAs, such as mirtrons and agotrons, due to the limitations of retroviral reverse transcriptases, which hinders the identification of biomarkers in these RNAs for specific characteristics, diseases, or conditions.
Innovation Solution
Employing thermostable group II intron reverse transcriptases, which are part of non-LTR-retroelement reverse transcriptases, to obtain full-length sequence reads of structured RNAs, allowing for the identification and analysis of biomarkers in Full-Length Excised Intron RNAs (FLEXI RNAs) using RNA sequencing methods.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If retroviral reverse transcriptases are used for RNA sequencing, then the sequencing process can be performed, but full-length end-to-end sequence reads of structured spliceosomal intron RNAs cannot be obtained
Solution Approach 1:
The patent changes the key parameter of reverse transcriptase selection from retroviral RT to thermostable group II intron RT (TGIRT). This parameter change enables the enzyme to handle structured RNAs like mirtrons and agotrons that were previously inaccessible, achieving full-length end-to-end sequence reads where conventional methods failed.
Solution Approach 2:
The patent introduces TGIRT as an intermediary enzyme that bridges the gap between available sequencing technologies and the need to sequence structured intron RNAs. This intermediary enzyme possesses unique properties (thermostability, ability to traverse secondary structures) that enable it to perform reverse transcription on RNAs that conventional retroviral RTs cannot process.
2Productivity
If conventional RNA-seq methods are used, then sequencing can be performed, but biomarkers in structured intron RNAs cannot be identified
Solution Approach 1:
By changing the reverse transcriptase parameter to TGIRT, the method recovers information about FLEXI RNAs (full-length excised intron RNAs) that was previously lost. This enables identification of biomarkers in structured intron RNAs while maintaining sequencing productivity.
Solution Approach 2:
The patent extracts and sequences previously inaccessible FLEXI RNAs from the total RNA population. By using TGIRT, the method isolates and sequences structured intron RNAs that contain biomarker information, extracting this valuable information from the complex RNA mixture without losing productivity.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables the detection and analysis of FLEXI RNAs as biomarkers for specific traits, diseases, or conditions, providing a biosignature for diagnostic, predictive, or prognostic purposes, and facilitating the understanding of gene expression and alternative splicing.
Implementation Method 1
Employing thermostable group II intron reverse transcriptases, which are part of non-LTR-retroelement reverse transcriptases, to obtain full-length sequence reads of structured RNAs
Data Source
AI summary
Disclosed herein are methods and compositions related to determining one or more biomarkers in Full-Length Excised Linear Intron RNAs (FLEXI RNAs) and Intron RNA fragments. These FLEXI RNAs and Intron RNA fragments can be indicative of a specific characteristic. trait. disease. disorder or condition. FLEXI RNAs and Intron RNA fragments can be used to establish a predictive bio-marker. a diagnostic biomarker, a prognostic biomarker, or a biomarker that relates to drug interaction, drug response, or to a heritable condition. These biomarkers can then be used to treat. monitor. or inform patients.